甘氨酸对经烧伤大鼠血清干预的大鼠心肌细胞的作用及其机制  

Effects and mechanism of glycine on rat cardiomyocytes pretreated with serum from burned rats

作　　者：吕尚军 闫尊东 范荣辉[3] 彭曦 Lyu Shangjun;Yan Zundong;Fan Ronghui;Peng Xi(Department of Burns and Plastic Surgery,Shaanxi Provincial Armed Police Corps Hospital,Xi'an 710086,China;Xi'an Beilin Tiansi Comprehensive Outpatient Department,Xi'an 710003,China;Department of Burns and Plastic Surgery and Medical Cosmetic Surgery,Shaanxi Provincial People's Hospital,Xi'an 710068,China;Clinical Medical Research Center,the First Affiliated Hospital of Army Medical University(the Third Military Medical University),Chongqing 400038,China)

机构地区：[1]武警陕西省总队医院烧伤整形科,西安710086 [2]西安碑林天嗣综合门诊部,西安710003 [3]陕西省人民医院烧伤整形医学美容外科,西安710068 [4]陆军军医大学(第三军医大学)第一附属医院临床医学研究中心,重庆400038

出　　处：《中华烧伤与创面修复杂志》2023年第5期434-442,共9页Chinese Journal of Burns And Wounds

基　　金：国家自然科学基金面上项目(81971838)。

摘　　要：目的探讨甘氨酸对经烧伤大鼠血清(以下简称烧伤血清)干预的大鼠心肌细胞的作用及其机制。方法采用实验研究方法。取30只7~8周龄雌雄各半Wistar大鼠,其中10只用于制备正常大鼠血清(以下简称正常血清),将另20只造成30%体表总面积Ⅲ度烧伤后制备烧伤血清;从180只1~3 d龄雌雄不拘Wistar大鼠心尖组织中分离培养原代心肌细胞用于后续实验。按随机数字表法(分组方法下同)将细胞分为用相应血清处理的正常血清组、烧伤血清组,于处理1、3、6、9、12 h进行锥虫蓝染色检测细胞存活率;将细胞分为用烧伤血清处理6 h后常规培养30 min的单纯烧伤血清组和用烧伤血清处理6 h后加相应终物质的量浓度甘氨酸培养30 min的0.4 mmol/L甘氨酸组、0.8 mmol/L甘氨酸组、1.2 mmol/L甘氨酸组、1.6 mmol/L甘氨酸组、2.0 mmol/L甘氨酸组,即干预6.5 h,同前检测细胞存活率。将细胞分为正常血清组、单纯烧伤血清组、0.8 mmol/L甘氨酸组、1.2 mmol/L甘氨酸组、1.6 mmol/L甘氨酸组,分别同前干预6.5 h,采用高效液相色谱法检测腺苷一磷酸(AMP)和ATP含量并计算AMP/ATP比值,采用蛋白质印迹法检测磷酸化哺乳动物雷帕霉素靶蛋白复合物1(p-mTORC1)、磷酸化p70核糖体蛋白S6激酶(p-p70 S6K)、磷酸化真核翻译起始因子4E结合蛋白1(p-4E-BP1)、磷酸化AMP活化蛋白激酶(p-AMPK)蛋白表达。将细胞分为同前干预的正常血清组、单纯烧伤血清组、0.8 mmol/L甘氨酸组和用烧伤血清处理后加2种试剂培养的0.8 mmol/L甘氨酸+25 ng/mL雷帕霉素组,于处理1、3、6 h行相应培养30 min,即干预1.5、3.5、6.5 h,采用免疫荧光法检测热休克蛋白70(HSP70)、金属硫蛋白(MT)和微管蛋白表达并观察干预6.5 h微管形态。各时间点样本数均为10。对数据行析因设计方差分析、单因素方差分析、LSD-t检验、LSD检验及Bonferroni校正。结果处理1、3、6、9、12 h,烧伤血清组细胞存活Objective To investigate the effect and mechanism of glycine on rat cardiomyocytes pretreated with serum from burned rats(hereinafter referred to as burn serum).Methods Experimental research methods were adopted.Thirty gender equally balanced Wistar rats aged 7 to 8 weeks were collected,10 of which were used to prepare normal rat serum(hereinafter referred to as normal serum),and the other 20 were inflicted with full-thickness burn of 30%total body surface area to prepare burn serum.Primary cardiomyocytes were isolated and cultured from the apical tissue of 180 Wistar rats aged 1 to 3 days by either gender for follow-up experiments.Cells were divided into normal serum group and burn serum group treated with corresponding serum according to the random number table(the same grouping method below).Trypanosoma blue staining was performed at post treatment hour(PTH)1,3,6,9,and 12 to detect the cell survival rate.Cells were divided into burn serum alone group treated with burn serum for 6 h followed by routine culture of 30 min and 0.4 mmol/L glycine group,0.8 mmol/L glycine group,1.2 mmol/L glycine group,1.6 mmol/L glycine group,and 2.0 mmol/L glycine group treated with burn serum for 6 h followed by culture of 30 min with corresponding final molarity of glycine,i.e.,at post intervention hour(PIH)6.5,the cell survival rate was detected as before.Cells were divided into normal serum group,burn serum alone group,0.8 mmol/L glycine group,1.2 mmol/L glycine group,and 1.6 mmol/L glycine group,with the same intervention of 6.5 h as before,respectively.The content of adenosine monophosphate(AMP)and adenosine triphosphate(ATP)was detected by high performance liquid chromatography,and the AMP/ATP ratio was calculated.The protein expressions of phosphorylated mammalian target of rapamycin complex 1(p-mTORC1),phosphorylated p70 ribosomal protein S6 kinase(p-p70 S6K),phosphorylated eukaryotic translation initiation factor 4E-binding protein 1(p-4E-BP1),and phosphorylated AMP-activated protein kinase(p-AMPK)were detected by Wester

关 键 词：烧伤 甘氨酸 肌细胞 心脏 能量代谢 微管 细胞保护 哺乳动物雷帕霉素靶蛋白信号通路 

分 类 号：R644[医药卫生—外科学]